Modified amino acids for drug delivery

ABSTRACT

The present invention relates to an oral delivery system, and in particular to modified amino acids or peptides for use as a delivery system of sensitive agents such as bioactive peptides. The modified amino acids or peptides can form non-covalent mixtures or microspheres with active biological agents. These mixtures or microspheres are suitable for oral administration of biologically active agents to animals. Methods for the preparation of such amino acids and peptides are also disclosed.

This is a division of application Ser. No. 08/231,622, filed Apr. 22,1994 now U.S. Pat. No. 5,629,020.

The present invention relates to compositions suitable for drugdelivery, and in particular to compositions in which modified aminoacids or peptides are used as carriers for biologically active agentsincluding, but not limited, to bioactive peptides and the like. Themodified amino acids or peptides can form non-covalent mixtures ormicrospheres with biologically-active agents and are suitable for oraladministration to animals. Methods for the preparation and for theadministration of such compositions are also disclosed.

BACKGROUND OF THE INVENTION

Conventional means for delivering biologically-active agents, including,but not limited to, pharmaceutical and therapeutic agents to animalsoften are severely limited by chemical and physical barriers imposed bythe body. Oral delivery of many biologically-active agents would be theroute of choice if not for the presence of chemical and physico-chemicalbarriers such as extreme and Varying pH in the gastrointestinal (GI)tract, exposure to powerful digestive enzymes, and impermeability ofgastrointestinal membranes to the active ingredient. Among the numerouspharmacological agents which are not suitable for oral administrationare biologically-active peptides such as calcitonin and insulin.Examples of other compounds which are affected by the physico-chemicalbarriers are polysaccharides and mucopolysaccharides, including, but notlimited to, heparin, heparinoids, antibiotics and other organicsubstrates. These agents are rapidly destroyed in the gastrointestinaltract by acid hydrolysis, enzymes, or the like.

Prior methods for orally administering vulnerable pharmacological agentshave relied on co-administration of adjuvants (e.g., resorcinols andnon-ionic surfactants such as polyoxyethylene oleyl ether andn-hexadecyl polyethylene ether) to increase artificially thepermeability of the intestinal walls; and on co-administration ofenzymatic inhibitors (e.g., pancreatic trypsin inhibitor,diisopropylfluorophosphate (DFF) and trasylol) to avoid enzymaticdegradation. Liposomes have also been described as drug delivery systemsfor insulin and heparin. See, for instance, U.S. Pat. No. 4,239,754;Patel et al. (1976) FEBS Letters Vol. 62, page 60; and Hashimoto et al.(1979) Endocrinol. Japan, Vol. 26, page 337. The broader use of theaforementioned methods, however, as drug delivery systems are precludedfor reasons which include: (1) the use of toxic amounts of adjuvants orinhibitors; (2) the lack of suitable low MW cargoes; (3) the poorstability and inadequate shelf life of the systems; (4) difficulty inmanufacturing; and (5) the failure of the systems to protect the activeingredient; and (6) the failure of the systems to promote absorption ofthe active agent.

More recently, microspheres of artificial polymers, or proteinoids, ofmixed amino acids have been described for delivery of pharmaceuticals.For example, U.S. Pat. No. 4,925,673 describes such microspheres as wellas methods for their preparation and use. The proteinoid microspheres ofthe '673 patent are useful for encapsulating a number of active agents.

There is a need in the art for a simple and inexpensive delivery systemwhich is easily prepared and which can deliver a broad range ofbiologically-active agents.

SUMMARY OF THE INVENTION

Compositions for delivering biologically-active agents incorporatingmodified amino acids as carriers are provided. The compositionscomprise;

(A) at least one biologically active agent, and

(B) (a) at least one acylated amino acid;

(b) at least one peptide comprising at least one acylated amino acid; or

(c) a combination of (a) and (b);

wherein said acylated amino acid is acylated by

(i) a C₃-C₁₀ cycloalkyl acylating agent, said agent optionally beingsubstituted with C₁-C₇ alkyl, C₂-C₇ alkenyl, C₁-C₇ alkoxy, hydroxy,phenyl, phenoxy, or —CO₂R, wherein R is hydrogen, C₁-C₄ alkyl or C₂-C₄alkenyl; or

(ii) a C₃-C₁₀ cycloalkyl substituted C₁-C₆ alkyl acylating agent.

In an alternative embodiment, these compositions are used in oral dosageunit forms. The compositions or oral dosage unit forms can be orallyadministered to animals.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic illustration of the results of oral gavage testingin rats using calcitonin with cyclohexanoyl-(L)-leucine,cycloheptanoyl-(L)-leucine and 2-methylcyclohexanoyl-(L)-leucinecarriers.

FIG. 2 is a graphic illustration of the results of oral gavage testingin rats using calcitonin with cyclohexanoyl-(L)-arginine,cyclopentanoyl-(L)-arginine, and cyclohexanoyl-(L)-phenylglycinecarriers.

FIG. 3 is a graphic illustration of the results of oral gavage testingin rats using calcitonin with cyclohexanoyl-(L)-arginine,cyclohexanoyl-(L)-leucine, and cyclohexanoyl-(L)-tyrosine carriers.

FIG. 4 is a graphic illustration of the results of oral gavage testingin rats using calcitonin with cyclohexanoyl-(L)-leucine,cyclohexanoyl-(L)-glycine and cyclopropanoyl-(L)-leucine carriers.

FIG. 5 is a graphic illustration of the results of oral gavage testingin rats using calcitonin with cyclohexanoyl-(L)-leucine carrier.

FIGS. 6 and 7 are graphic illustrations of the results of oral gavagetesting in rats using heparin with cyclohexanoyl-(L)-leucine carrier.

FIG. 8 is a graphic illustration of the results of oral gavage testingin rats using heparin with cyclohexanoyl-(L)-arginine carrier.

FIGS. 9 and 10 are graphic illustrations of the results of intraduodenalinjection testing in rats using heparin with cyclohexanoyl-(L)-leucinecarrier.

FIGS. 11 and 12 are graphic illustrations of the results of oral gavagetesting in rats using low molecular weight heparin withcyclohexanoyl-(L)-leucine carrier.

FIG. 13 is a graphic illustration of the results of oral gavage testingin rats using disodium cromoglycate with cyclohexanoyl-(L)-leucinecarrier.

FIG. 14 is a graphic illustration of the results of oral gavage testingin rats using interferon α2b (rhIFN) withcyclohexanoyl-(L)-phenylglycine and cyclohexanoyl-(L)-arginine carriers.

FIG. 15 is a graphic illustration of the results of oral administrationtesting in monkeys using interferon α2b with cyclohexanoyl-phenylene andcyclohexanoyl-arginine carriers.

FIG. 16 is a graphic illustration of the results of oral gavage andintraduodenal injection testing in rats using interferon α2b andcyclohexanoyl-(L)-phenylglycine carrier.

FIG. 17 is a graphic illustration of the results of oral gavage testingin rats using interferon α2b and cyclohexanoyl-(L)-phenylglycinecarrier.

DETAILED DESCRIPTION OF THE INVENTION

Modified amino acids and peptides that include at least one modifiedamino acid may be used as carriers to deliver biologically-active agentssuch as peptides, mucopolysaccharides, carbohydrates, lipids, andpesticides. These carriers particularly are useful in facilitating thedelivery of orally sensitive biologically active agents. For example,hormones such as calcitonin, insulin and polysaccharides such asheparin, are not considered orally administrable for various reasons.Insulin, for example, is sensitive to the denaturing conditions of thegastrointestinal (GI) tract. Also, heparin, by virtue of its charge andhydrophilic nature, is not readily absorbed from the gut. In contrast tothe modified amino acids and peptides of the present invention,unmodified free amino acids provide inadequate protection againstdegradation in the GI tract for labile bioactive agents.

The compositions of the subject invention are useful for administeringbiologically-active agents to any animals such as birds; mammals, suchas primates and particularly humans; and insects.

The present invention, in several embodiments, uses readily availableand inexpensive starting materials, and provides a cost-effective methodfor preparing and isolating modified amino acids and peptides. Themethod is simple to perform and is amenable to industrial scale-up forcommercial production.

Biologically-active agents suitable for use with carriers disclosedherein include, but are not limited to, peptides, and particularly smallpeptide hormones, which by themselves pass slowly or not at all throughthe gastrointestinal mucosa and/or are susceptible to chemical cleavageby acids and enzymes in the gastrointestinal tract; polysaccharides andparticularly mixtures of mucopolysaccharides, carbohydrates; lipids; orany combination thereof. Examples include, but are not limited to, humangrowth hormone; bovine growth hormone; growth hormone releasing hormone;interferons; interleukin-I; insulin; heparin, and particularly lowmolecular weight heparin; calcitonin; erythropoietin; atrial natureticfactor; antigens; monoclonal antibodies; somatostatin;adrenocorticotropin; gonadotropin releasing hormone; oxytocin;vasopressin; vancomycin; cromylyn sodium; desferrioxamine (DFO); or anycombination thereof.

Additionally the carriers of the present invention can be used todeliver other active agents such as pesticides and the like.

An amino acid is any carboxylic acid having at least one free aminegroup and includes naturally occurring and synthetic amino acids. Thepreferred amino acids for use in the present invention are ∝-aminoacids, and most preferably are naturally occurring ∝-amino acids. Polyamino acids are either peptides or two or more amino acids linked by abond formed by other groups which can be linked, e.g., an ester,anhydride or an anhydride linkage. Special mention is made ofnon-naturally occurring poly amino acids and particularly non-naturallyoccurring hetero poly amino acids, i.e. of mixed amino acids.

Peptides are two or more amino acids joined by a peptide bond. Peptidescan vary in length from di-peptides with two amino acids to polypeptideswith several hundred amino acids. See, Walker, Chambers BiologicalDictionary, Cambridge, England: Chambers Cambridge, 1989, page 215.Special mention is made of non-naturally occurring peptides andparticularly non-naturally occurring peptides of mixed amino acids. Thepeptides most useful in the practice of the present invention includedi-peptides, tri-peptides, tetra-peptides, and penta-peptides. Thepreferred peptides are di-peptides, and tri-peptides. Peptides can behomo- or hetero- peptides and can include natural amino acids, syntheticamino acids, or any combination thereof.

Amino acids suitable fur use in the present invention are generally ofthe formula

wherein: R¹ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl;

R² is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl) phenyl, (C₂-C₁₀ alkenyl)phenyl, (C₁-C₁₀ alkyl) naphthyl, (C₂-C₁₀ alkenyl) naphthyl, phenyl(C₁-C₁₀ alkyl), phenyl (C₂-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl), ornaphthyl (C₂-C₁₀ alkenyl);

R² being optionally substituted with C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁-C₄alkoxy, —OH, —SH, —CO₂R³, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,heterocycle having 3-10 ring atoms wherein the hetero atom is one ormore of N, O, S, or any combination thereof, aryl, (C₁-C₁₀ alkaryl,ar(C₁-C₁₀ alkyl) or any combination thereof;

R² being optionally interrupted by oxygen, nitrogen, sulfur, or anycombination thereof; and

R³ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl.

The preferred naturally occurring amino acids for use in the presentinvention as amino acids or components of a peptide are alanine,arginine, asparagine, aspartic acid, citrulline, cysteine, cystine,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,ornithine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, hydroxy proline, γ-carboxyglutamate, phenylglycine, orO-phosphoserine. The preferred amino acids are arginine, leucine,lysine, phenylalanine, tyrosine, tryptophan, valine, and phenylglycine.

The preferred non-naturally occurring amino acids for use in the presentinvention are β-alanine, α-amino butyric acid, γ-amino butyric acid,γ-(aminophenyl) butyric acid, α-amino isobutyric acid, ε-amino caproicacid, 7-amino heptanoic acid, β-aspartic acid, aminobenzoic acid,aminophenyl acetic acid, aminophenyl butyric acid, γ-glutamic acid,cysteine (ACM), ε-lysine, ε-lysine (A-Fmoc), methionine sulfone,norleucine, norvaline, ornithine, d-ornithine, p-nitro-phenylalanine,hydroxy proline, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid andthioproline.

The amino acids or peptides are modified by acylating at least one freeamine group, with an acylating agent which reacts with at least one ofthe free amine groups present. Suitable, but non-limiting, examples ofacylating agents useful for modifying amino acids or peptide derivativesuseful in practicing the present invention include acylating agents, andparticularly acid chloride acylating agents, having the formula

wherein R⁸ is

(i) C₃-C₁₀ cycloalkyl, optionally substituted with C₁-C₇ alkyl, C₂-C₇alkenyl, C₁-C₇ alkoxy, hydroxy, phenyl, phenoxy, or —CO₂R⁹ wherein R⁹ ishydrogen, C₁-C₄ alkyl or C₂-C₄ alkenyl; or

(ii) C₃-C₁₀ cycloalkyl substituted C₁-C₆ alkyl; and

X is a leaving group. Preferably, R⁸ is cyclopropyl, cyclopentyl,cyclohexyl or cycloheptyl.

In a reaction in which the substrate molecule becomes cleaved, part ofit (the part not containing the carbon) is usually called the leavinggroup. See Advanced Organic Chemistry, 2d edition, Jerry March, NewYork: McGraw-Hill Book (1977). Typical leaving groups include, but arenot limited to, halogens such as chlorine, bromine and iodine.

Preferred acylating agents include, but are not limited to, acyl halidessuch as cyclohexanoyl chloride, cyclopentanoyl chloride, cycloheptanoylchloride and the like; and anhydrides, such as cyclohexanoic anhydride,cyclopentanoic anhydride, cycloheptanoic anhydride, cycloheptanoicanhydride, and the like. Most preferred acylating agents arecyclohexanoyl chloride, cyclopentanoyl chloride, and cycloheptanoylchloride.

Preferred acylated amino acids of the present invention have the formula

wherein:

R⁴ is

(i) C₃-C₁₀ cycloalkyl, optionally substituted with C₁-C₇ alkyl, C₂-C₇alkenyl, C₁-C₇ alkoxy, hydroxy, phenyl, phenoxy or —CO₂R⁷, wherein R⁷ ishydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl; or

(ii) C₁-C₆ alkyl substituted with C₃-C₁₀ cycloalkyl;

R⁵ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl;

R⁶ is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl) phenyl, (C₂-C₁₀ alkenyl)phenyl, (C₁-C₁₀ alkyl) naphthyl, (C₂-C₁₀ alkenyl) naphthyl, phenyl(C₁-C₁₀ alkyl), phenyl (C₂-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl) ornaphthyl (C₂-C₁₀ alkenyl);

R⁶ being optionally substituted with C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁-C₄alkoxy, —OH, —SH, —CO₂R⁷, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,heterocycle having 3-10 ring atoms wherein the hetero atom is one ormore of N, O, S or any combination thereof, aryl, (C₁-C₁₀ alk)aryl,ar(C₁-C₁₀ alkyl), or any combination thereof;

R⁶ being optionally interrupted by oxygen, nitrogen, sulfur, or anycombination thereof; and

R⁷ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl.

The modified amino acids of the present invention may be prepared byreacting single amino acids, mixtures of two or more amino acids, aminoacid esters, or amino acid amides, with an amine modifying agent whichreacts with free amino moieties present in the amino acids to formamides. Amino acids and amino acid esters are readily available from anumber of commercial sources such as Aldrich Chemical Co. (Milwaukee,Wis., USA); Sigma Chemical Co. (St. Louis, Mont, USA); and FlukaChemical Corp. (Ronkonkoma, N.Y., USA).

The modified amino acids can be readily prepared by methods known tothose skilled in the art. For example, the amino acids are dissolved inan aqueous alkaline solution of a metal hydroxide, e.g., sodium orpotassium hydroxide and the acylating agent added. The reaction time canrange from about 1 hour and about 4 hours, preferably about 2-2.5 hours.The temperature of the mixture is maintained at a temperature generallyranging between about 5° C. and about 70° C., preferably between about10° C. and about 50° C. The amount of alkali employed per equivalent ofNH₂ groups in the amino acids generally ranges between about 1.25 molesand about 3 moles, and is preferably between about 1.5 moles and about2.25 moles per equivalent of NH₂. The pH of the reaction solutiongenerally ranges between about pH 8 and about pH 13, and is preferablybetween about pH 10 and about pH 12. The amount of amino modifying agentemployed in relation to the quantity of amino acids is based on themoles of total free NH₂ in the amino acids. In general, the aminomodifying agent is employed in an amount ranging between about 0.5 andabout 2.5 mole equivalents, preferably between about 0.75 and about 1.25equivalents, per molar equivalent of total NH₂ groups in the aminoacids.

The modified amino acid formation reaction is quenched by adjusting thepH of the mixture with a suitable acid, e.g., concentrated hydrochloricacid, until the pH reaches between about 2 and about 3. The mixtureseparates on standing at room temperature to form a transparent upperlayer and a white or off-white precipitate. The upper layer is discardedand modified amino acids are collected by filtration or decantation. Thecrude modified amino acids are then mixed with water. Insolublematerials are removed by filtration and the filtrate is dried in vacuo.The yield of modified amino acids generally ranges between about 30 andabout 60%, and usually about 45%. The present invention alsocontemplates amino acids which have been modified by multiple acylation,e.g., diacylation or triacylation.

If desired, esters or amides of amino acids may be used to prepare themodified amino acids of the invention. The amino acid esters or amides,dissolved in a suitable organic solvent such as dimethylformamide orpyridine, are reacted with the amino modifying agent at a temperatureranging between about 5° C. and about 70° C., preferably about 25° C.,for a period ranging between about 7 and about 24 hours. The amount ofamino modifying agents used relative to the amino acid esters are thesame as described above for amino acids.

Thereafter, the reaction solvent is removed under negative pressure andoptionally the ester or amide functionality can be removed byhydrolyzing the modified amino acid ester with a suitable alkalinesolution, e.g., 1N sodium hydroxide, at a temperature ranging betweenabout 50° C. and about 80° C., preferably about 70° C., for a period oftime sufficient to hydrolyze off the ester group and form the modifiedamino acid having a free carboxyl group. The hydrolysis mixture is thencooled to room temperature and acidified, e.g., with an aqueous 25%hydrochloric acid solution, to a pH ranging between about 2 and about2.5. The modified amino acid precipitates out of solution and isrecovered by conventional means such as filtration or decantation.

The modified amino acids may be purified by acid precipitation,recrystallization or by fractionation on solid column supports.Fractionation may be performed on a suitable solid column supports suchas silica gel, alumina, using solvent mixtures such as aceticacid/butanol/water as the mobile phase; reverse phase column supportsusing trifluoroacetic acid/acetonitrile mixtures as the mobile phase;and ion exchange chromatography using water as the mobile phase. Themodified amino acids may also be purified by extraction with a loweralcohol such as methanol, butanol, or isopropanol to remove impuritiessuch as inorganic salts.

The modified amino acids of the present invention generally are solublein alkaline aqueous solution (pH≧9.0); partially soluble in ethanol,n-butanol and 1:1 (v/v) toluene/ethanol solution and insoluble inneutral water. The alkali metal salts, e.g., the sodium salt of thederitivatized amino acids are generally soluble in water at about a pHof 6-8.

Modified peptides may include one or more acylated amino acid. Althoughlinear modified peptides will generally include only one acylated aminoacid, other peptide configurations such as, but not limited to, branchedpeptides can include more than one acylated amino acid. Peptides can bepolymerized with the acylated amino acid(s) or can be acylated afterpolymerization. Special mention is made of compounds having the formula:

wherein A is Try, Leu, Arg, Trp, or Cit; and

optionally wherein if A is Try, Arg, Trp or Cit; A is acylated at 2 ormore functional groups.

Preferred compounds are those wherein A is Try; A is Tyr and is acylatedat 2 functional groups; A is Leu; A is Arg; A is Arg and is acylated at2 functional groups; A is Trp; A is Trp and is acylated at 2 functionalgroups; A is Cit; and A is Cit and is acylated at 2 functional groups.

Special mention is also made of compounds having the formula:

wherein A is Arg or Leu and B is Arg or Leu

wherein A is Arg or Leu; and

wherein if A is Arg, A is optionally acylated at 2 or more functionalgroups;

where A is Leu or phenylglycine;

wherein A is phenylglycine; and

wherein A is phenylglycine.

If the amino acid is multifunctional, i.e. has more than one —OH, —NH2or —SH group, then it may optionally be acylated at one or morefunctional groups to form, for example, an ester, amide, or thioesterlinkage.

In one embodiment, the modified amino acids or peptides may be useddirectly as a drug delivery carrier by simply mixing one or moremodified amino acids or peptides with the active ingredient prior toadministration. In an alternative embodiment, the modified amino acidsmay be used to form microspheres containing the active agent. Themodified amino acids or peptides of the invention are particularlyuseful for the oral administration of certain biologically-activeagents, e.g., small peptide hormones, which, by themselves, do not passor only pass slowly through the gastrointestinal mucosa and/or aresusceptible to chemical cleavage by acids and enzymes in thegastrointestinal tract.

If the modified amino acids or peptides are to be converted intomicrospheres such as proteinoid microspheres, the mixture is optionallyheated to a temperature ranging between about 20 and about 50° C.,preferably about 40° C., until the modified amino acid(s) dissolve. Thefinal solution contains between from about 1 mg and to about 2000 mg ofmodified amino acids or peptides per mL of solution, preferably betweenabout 1 and about 500 mg per mL. The concentration of active agent inthe final solution varies and is dependent on the required dosage fortreatment. When necessary, the exact concentration can be determined by,for example, reverse phase HPLC analysis.

When the modified amino acids or peptides are used to preparemicrospheres, another useful procedure is as follows: Modified aminoacids or peptides are dissolved in deionized water at a concentrationranging between about 75 and about 200 mg/ml, preferably about 100 mg/mlat a temperature between about 25° C. and about 60° C., preferably about40° C. Particulate matter remaining in the solution may be removed byconventional means such as filtration.

Thereafter, the modified amino acid or peptide solution, maintained at atemperature of about 40° C., is mixed 1:1 (V/V) with an aqueous acidsolution (also at about 40° C.) having an acid concentration rangingbetween about 0.05 N and about 2 N, preferably about 1.7 N. Theresulting mixture is further incubated at 40° C. for a period of timeeffective for microsphere formation, as observed by light microscopy. Inpracticing this invention, the preferred order of addition is to add themodified amino acid or peptide solution to the aqueous acid solution.

Suitable acids for microsphere formation include any acid which does not

(a) adversely effect the modified amino acids, or peptides e.g.,initiate or propagate chemical decomposition;

(b) interfere with microsphere formation;

(c) interfere with microsphere incorporation of the cargo; and

(d) adversely interact with the cargo.

Preferred acids for use in this invention include acetic acid, citricacid, hydrochloric acid, phosphoric acid, malic acid and maleic acid.

In practicing the invention, a microsphere stabilizing additive may beincorporated into the aqueous acid solution or into the modified aminoacid or protein solution prior to the microsphere formation process.With some drugs the presence of such additives promotes the stabilityand/or dispersibility of the microspheres in solution.

The stabilizing additives may be employed at a concentration rangingbetween about 0.1 and 5% (w/v), preferably about 0.5% (w/v). Suitable,but non-limiting, examples of microsphere stabilizing additives includegum acacia, gelatin, methyl cellulose, polyethylene glycol, andpolylysine. The preferred stabilizing additives are gum acacia, gelatinand methyl cellulose.

Under the above conditions, the modified amino acid molecules orpeptides form hollow or solid matrix type microspheres wherein the cargois distributed in a carrier matrix or capsule type microspheresencapsulating liquid or solid cargo. If the modified amino acid orpeptide microspheres are formed in the presence of a soluble material,e.g., a pharmaceutical agent in the aforementioned aqueous acidsolution, this material will be encapsulated within the microspheres. Inthis way, one can encapsulate pharmacologically active materials such aspeptides, proteins, and polysaccharides as well as charged organicmolecules, e.g., antimicrobial agents, which normally have poorbioavailability by the oral route. The amount of pharmaceutical agentwhich may be incorporated by the microsphere is dependent on a number offactors which include the concentration of agent in the solution, aswell as the affinity of the cargo for the carrier. The modified aminoacid or peptide microspheres of the invention do not alter thephysiological and biological properties of the active agent.Furthermore, the encapsulation process does not alter thepharmacological properties of the active agent. Any pharmacologicalagent can be incorporated within the amino acid microspheres. The systemis particularly advantageous for delivering chemical or biologicalagents which otherwise would be destroyed or rendered less effective byconditions encountered within the body of the animal to which it isadministered, before the microsphere reaches its target zone (i.e., thearea in which the contents of the microsphere are to be released) andpharmacological agents which are poorly absorbed in the gastrointestinaltract. The target zones can vary depending upon the drug employed.

The particle size of the microsphere plays an important role indetermining release of the active agent in the targeted area of thegastrointestinal tract. The preferred microspheres have diametersbetween about ≦0.1 microns and about 10 microns, preferably betweenabout 0.5 microns and about microns. The microspheres are sufficientlysmall to release effectively the active agent at the targeted areawithin the gastrointestinal tract such as, for example, between thestomach and the jejunum. Small microspheres can also be administeredparenterally by being suspended in an appropriate carrier fluid (e.g.,isotonic saline) and injected directly into the circulatory system,intramuscularly or subcutaneously. The mode of administration selectedwill vary, of course, depending upon the requirement of the active agentbeing administered. Large amino acid microspheres (>50 microns) tend tobe less effective as oral delivery systems.

The size of the microspheres formed by contacting modified amino acidsor peptides with water or an aqueous solution containing active agentscan be controlled by manipulating a variety of physical or chemicalparameters, such as the pH, osmolarity or ionic strength of theencapsulating solution, size of the ions in solution and by the choiceof acid used in the encapsulating process.

Typically, the pharmacological compositions of the present invention areprepared by mixing an aqueous solution of the carrier with an aqueoussolution of the active ingredient, just prior to administration.Alternatively, the carrier and biologically active ingredient can beadmixed during the manufacturing process. The solutions may optionallycontain additives such as phosphate buffer salts, citric acid, aceticacid, gelatin and gum acacia.

In practicing the invention, stabilizing additives may be incorporatedinto the carrier solution. With some drugs, the presence of suchadditives promotes the stability and dispersibility of the agent insolution.

The stabilizing additives may be employed at a concentration rangingbetween about 0.1 and 5% (W/V), preferably about 0.5% (W/V). Suitable,but non-limiting, examples of stabilizing additives include gum acacia,gelatin, methyl cellulose, polyethylene glycol, and polylysine. Thepreferred stabilizing additives are gum acacia, gelatin and methylcellulose.

The amount of active agent in the composition typically is apharmacologically or biologically effective amount. However, the amountcan be less than a pharmacologically or biologically effective amountwhen the composition is used in a dosage unit form, such as a capsule, atablet or a liquid, because the dosage unit form may contain amultiplicity of carrier/biologically-active agent compositions or maycontain a divided pharmacologically or biologically effective amount.The total effective amounts will be administered by cumulative unitscontaining, in total, pharmacologically or biologically active amountsof biologically-active agent.

The total amount of biologically-active agent to be used can bedetermined by those skilled in the art. However, it has surprisinglybeen found that with certain biologically-active agents, such ascalcitonin, the use of the presently disclosed carriers providesextremely efficient delivery. Therefore, lower amounts ofbiologically-active agent than those used in prior dosage unit forms ordelivery systems can be administered to the subject, while stillachieving the same blood levels and therapeutic effects.

The amount of carrier in the present composition is a delivery effectiveamount and can be determined for any particular carrier orbiologically-active agent by methods known to those skilled in the art.

Dosage unit forms can also include any of excipients; diluents;disintegrants; lubricants; plasticizers; colorants; and dosing vehicles,including, but not limited to water, 1,2-propane diol, ethanol, oliveoil, or any combination thereof.

Administration of the present compositions or dosage unit formspreferably is oral or by intraduodenal injection.

EXAMPLES

The invention will now be illustrated in the following non-limitingexamples which are illustrative of the invention but are not intended tolimit the scope of the invention.

Example 1

PREPARATION OF N-CYCLOHEXANOYL-(L)-TYROSINE.

(L)-Tyrosine (61.6 g., 0.34 mole) was dissolved in 190 mL of 2N sodiumhydroxide. Cyclohexanoyl chloride (49.32 mL, 0.34 mole) was addeddropwise to the mixture. Additional aqueous 2N sodium hydroxide wasadded and the reaction mixture was allowed to stir at room temperaturefor 2 hours. The mixture was then acidified to pH 9.5 with aqueous (4:1)hydrochloric acid. A precipitate formed which was separated by vacuumfiltration. The solids were dissolved in 2N sodium hydroxide and driedby lyophilization to furnish 33.5 g of N,O-dicyclohexanoyl-(L)-tyrosine.The product was purified by column chromatography on silica gel usingbutanol/acetic acid/water as the eluent system. The pure product was awhite solid.

1. Mass Spectrum: M+23 m/e 314.

2. ¹H NMR (300 MHz,DMSO-d6): d=6.8 (d, 2H); 6.4 (d,2H); 4.4 (m, 1H); 2.5(ddd,2H); 2.0 (m,2H); 1.6 (m,10H); 1.2(m, 10H).

3. IR (KBr) cm−1: 3350, 2900, 2850, 1600, 1520, 1450, 1400, 1300.

Example 2

PREPARATION OF N-CYCLOHEXANOYL-(L)-ARGININE.

(L)-Arginine (103.2 g., 0.6 mole) was dissolved in 600 mL of 2N sodiumhydroxide. Cyclohexanoyl chloride (87 mL, 0.6 mole) was added dropwiseto the mixture. The reaction mixture was maintained at 50° C. for 2hours. The mixture was then cooled to room temperature and acidified topH 2.3 with aqueous (4:1) hydrochloric acid. The precipitate whichformed was separated by decantation. The solids were dissolved in 2Nsodium hydroxide and dried by Iyophilization to furnish 64.1 g of crudeN-cyclohexanoyl-(L)-arginine. The product was purified by columnchromatography on silica gel/using butanol/acetic acid/water as theeluent system. The products isolated were N-cyclohexanoyl-(L)-arginineand N(α)-N(γ)-dicyclohexanoyl-(L)-arginine.

N-cyclohexanoyl-(L)-arginine:

1. Mass Spectrum: M+1 m/e 395.

2. ¹H NMR (300 MHz, DMSO-d6): ppm δ=8.75(br, 1H); 7.6 (br, 5H); 4.0 (m,1H); 3.05 (m, 2H); 2.15 (m, 1H); 1.1-1.5 (br.m, 14H).

N(α),N(γ)-dicyclohexanoyl-(L)-arginine:

1. Mass Spectrum: M+1 m/e 285.

2. ¹H NMR: (300 MHz, DMSO-d6): d=2.0 (m, 3H); 1.8-1.4 (br. m, 17H);1.3-1.0 (br. m, 20H)

Example 3

PREPARATION OF N-CYCLOHEXANOYL-(L)-CITRULLINE.

L-Citrulline (35.2 g., 0.2 mole) was dissolved in 200 mL of 2N sodiumhydroxide. Cyclohexanoyl chloride (29 mL, 0.2 mole) was added dropwiseto the mixture. The reaction mixture was maintained at about 25° C. for1 hour. The mixture was then acidified to pH 2.6 with aqueous (4:1)hydrochloric acid. The precipitate which formed was separated bydecantation. The solids were dissolved in 2N sodium hydroxide to pH 6.5and dried by lyophilization to furnish 44.2 g ofN-cyclohexanoyl-(L)-citrulline. The product was a white solid.

1. Mass Spectrum: M+23 m/e 308.

2. ¹H NMR (300 MHz,DMSO-d6): d=4.1 (dd, 1H); 2.9 (t, 2H); 2.1 (m,2H);1.6-1.2 (br.m, 14H).

3. IR (KBr) cm−1: 3400, 3300, 2950, 2850, 1700, 1650, 1600, 1450, 1400cm−1.

Example 4

PREPARATION OF N-CYCLOPENTANOYL-(L)-ARGININE.

(L)-Arginine (32.8 g., 0.19 moles) was dissolved in 188 mL of 2N sodiumhydroxide. Cyclopentanoyl chloride (22.9 mL, 0.19 moles) were addeddropwise to the mixture. The reaction mixture was maintained at about25° C. for 2 hours. The mixture was then acidified to pH 1.5 withaqueous (4:1) hydrochloric acid. The precipitate which formed wasseparated by decantation. The solids were dissolved in 2N sodiumhydroxide to pH 7.5 and dried by lyophilization to furnish 67.4 g ofN-cyclopentanoyl-(L)-arginine. The product was a white solid. MassSpectrum: M+1 m/e 271.

Example 5

PREPARATION OF N-CYCLOHEXANOYL-(t)-ARGININE.

(t)-Arginine (14.2 g., 0.1 mole) was dissolved in 100 mL of 2N sodiumhydroxide. Cyclohexanoyl chloride (13 mL, 0.098 mole) was added dropwiseto the mixture. The reaction mixture was maintained at 25° C. for 2hours. The mixture was then cooled to room temperature and acidified topH 6.6 with aqueous (4:1) hydrochloric acid. The white precipitate whichformed was separated by decantation. The solids were dissolved in aminimum of 2N sodium hydroxide. The product, a white solid, (11.6 g,49%) was isolated by lowering the pH of the purified by acidificationwith aqueous (4:1) hydrochloric acid to a pH of about 7-9.

1. Mass Spectrum: M+1 m/e 2423

2. ¹H NMR (300 MHz, D₂O): ppm δ=4.9 (s, 1H); 2.2 (m, 1H); 1.7-1.4 (m,5H); 1.3-1.0 (m, 5H); 0.8 (s, 9H).

3. IR (KBr) cm−1: 3350, 2950, 2850, 1550, 1500, 1400 cm⁻¹

Following the procedure of Example 1 the following amino acids andpeptides have been synthesized:

cyclohexanoyl-Ala, m-(cyclohexanolyamino)benzoic acid,p-(cyclohexanoylamino)benzoic acid, 4-(cyclohexanoyl-amino)butyric acid,6-(cyclohexanoylamino)hexanoic acid, cyclohexanoylanthranilic acid,cyclohexanoyl-Arg-Leu, cyclohexanoyl-Asp, isatoicanhydride-Asp,cyclohexanoyl-Glu, cyclohexanoyl-Gly, cyclohexanoyl-Gly-Arg,cyclohexanoyl-lle, cyclohexanoyl-Leu, cyclopentanoyl-Leu,cyclopropanoyl-Leu, 3-methycyclohexanoyl-Leu, 2-methycyclohexanoyl-Leu,4-methycyclohexanoyl-Leu, cyclohexanoyl-(D)-Leu, cyclohexanoyl-(t)-Leu,cyclohexanoyl-Leu-Arg, cyclohexanoyl-Leu-Leu,cyclohexanoyl-(D)-Leu-(L)-Leu, cyclohexanoyl-Leu-Lys-Val,cyclohexanoyl-Lys, cyclohexanoyl-Orn, cyclohexanoyl-Phe,cycloheptanoyl-Phg, cyclohexylpropanoyl-Phg, cyclohexanoyl-Phg,cyclopentanoyl-Phg, cyclopropanoyl-Phg, 4-methycyclohexanoyl-Phg,cyclohexanoyl-(D)-Phg, cyclohexanoyl-Tio, cyclohexanoyl-Trp,cyclohexanoyl-Tyr-Leu, cyclohexanoyl-Val, cyclopentanoyl-Val,cyclohexanoyl-Val-Val, cycloheptanoyl-Leu, and cyclohexylpropanoyl-Leu.

Example 6

PREPARATION OF CALCITONIN DOSING SOLUTIONS:

In a test tube 400 mg of cyclohexanoyl-(L)-leucine was added to 2.9 mlof 15% ethanol. The solution was stirred and NaOH (1.0 N) was added toraise the pH to 7.2. Water was added to bring the total volume to 4.0mL. The sample had a carrier concentration of 200 mg/mL. Calcitonin (10μg) was added to the solution. The total calcitonin concentration was2.5 μg/mL.

Following a similar procedure a second solution having 400 mg ofcycloheptanoyl-(L)-leucine as the carrier and a third solution having2-methylcyclohexanoyl-(L)-leucine as the carrier were prepared. Eachsolution had a calcitonin concentration of 2.5 μg/mL.

Example 7

CALCITONIN In Vivo EXPERIMENTS IN RATS

For each sample a group of fasted rats were anesthetized. The rats wereadministered, by oral gavage or by intraduodenal injection, one of thecalcitonin/carrier dosages prepared in Example 6. The calcitoninconcentration in each sample was 2.5 μg/ml. Each rat was administered adosage of four (4) mL/kg each. Blood samples were collected seriallyfrom the tail artery. Serum calcium was determined by testing with aDemand™ Calcium Kit (available from Sigma Chemical Company, St. Louis,Mo., USA). The results of the test are illustrated in FIG. 1.

Example 8

Three samples having 400 mg/kg of cyclohexanoyl-(L)-arginine and 10μg/kg of calcitonin, 400 mg/kg of cyclopentanoyl-(L)-arginine and 10μg/kg of calcitonin, 400 mg/kg of cyclohexanoyl-(L)-phenylglycine and 10μg/kg of calcitonin, respectively were prepared. The samples were givento fasted rats as described in Example 7. The results of the test areillustrated in FIG. 2.

Example 9

A sample having a mixture of 266 mg/kg of cyclohexanoyl-(L)-arginine 266mg/kg of cyclohexanoyl-(L)-leucine 266 mg/kg ofcyclohexan-oyl-(L)-tyrosine and 10 μg/kg of calcitonin, was prepared.The sample was given to fasted rats as described in Example 7. Theresults of the test are illustrated in FIG. 3.

Example 10

A series of samples having 400 mg/kg of cyclohexanoyl-(L)-leucine and 3μg/kg of calcitonin, 400 mg/kg of cyclohexanoyl-(L)-glycine and 3 μg/kgof calcitonin, 400 mg/kg of cyclopropanoyl-(L)-leucine and 3 μg/kg ofcalcitonin, respectively were prepared. The samples were given to fastedrats as described in Example 7. The results of the test are illustratedin FIG. 4.

Example 11

Two samples were prepared, having 400 mg/kg of cyclohexanoyl-(L)-leucineand 10 μg/kg of calcitonin, and cyclohexanoyl-(L)-leucine and 3 μg/kg ofcalcitonin, respectively. The samples were given to fasted rats asdescribed in Example 7. The results of the test are illustrated in FIG.5.

Example 12

PREPARATION OF HEPARIN DOSING SOLUTIONS:

Following the general procedure published by Santiago, N. in Proc. Int.Symp. Control Rel. Bioact. Mat., Vol. 19. pages 514-515, (1992) theheparin samples were prepared. In a test tube 900 mg ofcyclohexanoyl-(L)-leucine was added to 4.5. mL of water. Heparin (74.7mg) was dissolved in 4.5 mL of a solution of 1.7 N citric acid and 0.5%gum arabic. The solutions were warmed to about 40° C. and mixed. Thesample had a carrier concentration of 100 mg/mL. The heparinconcentration was 8.3 mg/mL.

Following a similar procedure a second sample having 900 mg ofcyclohexanoyl-(L)-leucine and heparin (150 mg) was prepared. The heparinconcentration was 16.7 mg/mL.

Example 13

HEPARIN In Vivo EXPERIMENTS IN RATS

For each sample a group of fasted rats were anesthetized. The rats wereadministered, by oral gavage, one of the heparin/carrier dosagesprepared in Example 11. The heparin concentration in the samples were8.3 and 16.7 mg/ml respectively. Each rat was administered a dosage ofabout three (3) mL/kg each. Blood samples were collected serially fromthe tail artery. Heparin activity was determined by utilizing theactivated partial thromboplastin time (APTT) according to the method ofHenry, J. B., Clinical Diagnosis and Management by Laboratory Methods;Philadelphia, Pa.; W B Saunders (1979). The results of the test areillustrated in FIG. 6.

Example 14

Two samples were prepared, having 600 mg/kg of cyclohexanoyl-(L)-leucineand 50 mg/kg of heparin and 600 mg/kg of cyclohexanoyl-(L)-leucine and100 mg/kg of heparin, respectively. The samples were given to fastedrats as described in Example 13. The results of the test are illustratedin FIG. 7.

Example 15

Two samples were prepared, having 100 mg/kg ofcyclohexanoyl-(L)-arginine and 100 mg/kg of heparin and 600 mg/kg ofcyclohexanoyl-(L)-arginine and 100 mg/kg of heparin, respectively. Thesamples were given to fasted rats as described in Example 1 2. Theresults of the test are illustrated in FIG. 8.

Example 16

A sample having 300 mg/kg of cyclohexanoyl-(L)-leucine and 25 mg/kg ofheparin was prepared. The sample was given to rats by intraduodenalinjection. As a comparison heparin, at a dose of 25 mg/kg wasadministered by intraduodenal injection. The results of the test areillustrated graphically in FIG. 9.

Example 17

A sample having 300 mg/kg of cyclohexanoyl-(L)-leucine and 50 mg/kg ofheparin was prepared. The sample was given to rats by intraduodenalinjection. As a comparison cyclohexanoyl-(L)-leucine without any heparinwas administered by intraduodenal injection. After 30 minutes this wasfollowed by a dose of heparin, 50 mg/kg administered by intraduodenalinjection. A second comparison, a dose of heparin alone, 50 mg/kg, wasalso administered by intraduodenal injection. The results of the testare illustrated graphically in FIG. 10.

Example 18

PREPARATION OF LOW MOLECULAR WEIGHT HEPARIN SAMPLES

Samples containing low molecular weight heparin were prepared asdescribed in Example 12.

Example 19

LOW MOLECULAR WEIGHT HEPARIN In Vivo EXPERIMENTS IN RATS

Samples containing low molecular weight heparin (LMWH) andcyclohexanoyl-(L)-leucine as described in Example 19 were prepared andadministered, by oral gavage, to a group of fasted rats. Blood sampleswere collected serially from the tail artery. Low molecular weightheparin (LMWH) was determined in plasma samples. The plasma level wasmeasured with an antiFactor Xa assay kit available from ChromogenixA.B., Sweden. The results of the test are illustrated in FIG. 11.

Example 20

A sample having 300 mg/kg of cyclohexanoyl-(L)-leucine and 8000 IU/kglow molecular weight heparin was prepared. The sample was given tofasted rats as described in Example 20. The results of the test areillustrated in FIG. 12.

In Vivo EVALUATION OF CROMOGLYCOLATE PREPARATIONS IN RATS

Example 21

Following the procedures described herein samples containing thecarriers of the subject invention and disodium cromoglycolate wereprepared. The sample, in 0.85N citric acid and 0.5% acacia, contained400 mg/kg of cyclohexanoyl-(L)-leucine and 50 mg/kg of disodiumcromoglycate (DSCG). The pH of this sample was 7.1. A second sample wasprepared at a pH of 4.6. The animals were administered the samples byoral gavage. As a comparison the DSCG was delivered in water, pH 7.2,and in citric acid, pH 3.7. The delivery was evaluated by using theprocedure described by A. Yoshimi in Pharmcobio-Dyn., 15, pages 681-686,(1992). The results of the tests are illustrated in FIG. 13.

In Vivo EVALUATION OF INTERFERON PREPARATIONS IN RATS

Example 22

Following the procedures described herein samples containing thecarriers of the subject invention, in a Trizma® hydrochloride buffersolution (Tris-HCl) at a pH of about 7-8, and interferon α2b wereprepared. The animals were administered the drug by oral gavage. Thedelivery was evaluated by using an ELISA assay for human interferon α.

Two samples having 800 mg/kg of cyclohexanoyl-(L)-phenylglycine in abuffered solution and 1000 μg/kg of interferon α2b and 800 mg/kgcyclohexanoyl-(L)-arginine in a buffered solution and 1000 μg/kg ofinterferon α2b were prepared. The samples were given to fasted rats byoral gavage. The results of the test are illustrated in FIG. 14.

Example 23

Two samples having 800 mg/kg of cyclohexanoyl-(L)-phenylglycine in abuffered solution and 1000 μg/kg of interferon α2b andcyclohexanoyl-(L)-arginine in a buffered solution and 1000 μg/kg ofinterferon α2b were prepared. The samples were orally administered tomonkeys. The results of the test are illustrated in FIG. 15.

Example 24

A sample having 400 mg/kg of cyclohexanoyl-(L)-phenylglycine in abuffered solution and 500 μg/kg of interferon α2b was prepared. Thesample was given to fasted rats by oral gavage. The sample was alsogiven to a second group of rats by intraduodenal injection. The resultsof the test are illustrated in FIG. 16.

Example 25

Three samples having 400 mg/kg of cyclohexanoyl-(L)-phenylglycine in abuffered solution with 1000 μg/kg of interferon α2b, 500 μg/kg ofinterfer-on α2b and 250 μg/kg of interferon α2b were prepared. Thesamples were given to fasted rats by oral gavage. The results of thetest are illustrated in FIG. 17.

All patents, patent applications, literature publications and testmethods cited herein are hereby incorporated by reference.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed disclosure. Allsuch modifications are within the full intended scope of the appendedclaims.

What is claimed is:
 1. A compound having the formula

wherein A is arginine, or citrulline.
 2. A compound according to claim1, wherein A is acylated at two or more functional groups.
 3. A compoundaccording to claim 1, wherein A is arginine.
 4. A compound according toclaim 2, wherein A is arginine.
 5. A compound according to claim 2,wherein A is tryptophan.
 6. A compound according to claim 1, wherein Ais citrulline.
 7. A compound according to claim 2, wherein A iscitrulline.
 8. A compound having the formula

wherein A and B independently are arginine or leucine.
 9. A compoundaccording to claim 8, wherein if A, B or A and B are arginine , A, B, orA and B are acylated at two or more functional groups.
 10. A compoundhaving the formula

wherein A is arginine.
 11. A compound according to claim 10, wherein Ais acylated at two or more functional groups.
 12. A compound having theformula

wherein A is leucine.
 13. A compound having the formula

wherein A is phenylglycine.
 14. A compound having the formula

wherein A is phenylglycine.